CA2024804A1 - Prostaglandin-containing liposome preparations - Google Patents
Prostaglandin-containing liposome preparationsInfo
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- CA2024804A1 CA2024804A1 CA002024804A CA2024804A CA2024804A1 CA 2024804 A1 CA2024804 A1 CA 2024804A1 CA 002024804 A CA002024804 A CA 002024804A CA 2024804 A CA2024804 A CA 2024804A CA 2024804 A1 CA2024804 A1 CA 2024804A1
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- Prior art keywords
- prostaglandin
- lipid
- containing liposome
- liposome preparation
- liposome
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
- A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
- A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/557—Eicosanoids, e.g. leukotrienes or prostaglandins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- Organic Chemistry (AREA)
- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
ABSTRACT
The prostaglandin-containing liposome preparations which are characterized in that positive-charged lipid(s) is (are) incorporated in lipid thin membrane therein are discussed, and according to the present invention, release of prosta-glandins from liposomes can be suppressed.
The prostaglandin-containing liposome preparations which are characterized in that positive-charged lipid(s) is (are) incorporated in lipid thin membrane therein are discussed, and according to the present invention, release of prosta-glandins from liposomes can be suppressed.
Description
~f~
SPECIFICATION
Prostaglandin-containing Liposome Preparations FIELD OF THE INVENTION
This invention relates to prostaglandin-containing lipo-some preparations in which release of prostaglandin (herein-aEter referred to as PG) is suppressed.
BACKGRO~ND OF THE INVENTION
Since PGs possess various physiological actions such as vasodilating action, peripheral circulation-improving action, antihypertensive action, antilipolytic action and sodium diuretic action, they have been applied to pharmaceuticals.
In app]ying PGs to pharmaceuticals, there are problems that PGs metabolize into inactive substances easily in ]iving bodies and that they have little selectivity of focuses.
Therefore, preparations of PGs generally require fre-quent administrations, which is likely to give pain to pa-tients and also side-effects to tissues other than the target tissues. As a means to solve these problems, there have been provided preparations in which PGs are contained in a liposome.
Even in ~hese preparations of PGs-containing liposome, however, the stability thereof is not satisfactory and there is still a problem that PGs, which were once contained in the liposome get released.
SUMMARY OF THE INVENTION
The object of this invention is to provide PGs-containing liposome preparations in which release of PGs therefrom is suppressed.
The present inventors conducted various studies on the way to suppress release of PGs from liposome in preparations in which PGs are con-tained in liposome, and found that the problem could be solved by incorporating positive-charged lipid(s) in lipid membrane, which resulted in the comple-tion of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. la and lb show the profiles of gel filtration of the liposomes a - d, which indicate release of PGEl at pH
7.4. Fig. 2 shows the profiles of gel filtration of the liposomes a and d, which show release of PGEl in the presence of albumin.
DETAILED DESCRIPTION OF THE INVENTION
That is, this invention relates to prostaglandin-containing liposome preparations which are characteri~ed in that positive-charged lipid(s) is(are) incorporated in lipid membrane.
In this invention, fat corpuscles (liposome) have a structure of multiple concentric double layers, a single layer or multiple layers and are capable of entrapping va-rious proteins or other physiologically active substances in the gaps therein. The preparations of the present invention can be prepared by, for e~ample, bringing thin membranes made :' of phospholipid(s) or other kinds of lipids and positive-charged lipid(s) into contact with a solution containing PG and allowing PG to be entrapped in the lipid membrane and the gaps of the liposome.
As the lipid forming said liposomes, there may be mentioned phospholipids, glycolipids, derived lipids and the like. As the phospholipid, any phospholipid can be used as long as it is physiologically acceptable and can be matabo-lized. For example, use can be made of phosphatidyl choline, phosphatidyl serine, phosphatidic acid, phosphatidyl glyce-rin, phosphatidyl ethanolamine, phosphatidyl inositol, sphin-gomyelin, dicetyl phosphate, lysophosphatidyl choline (lyso-lecithine), mixtures thereof such as soybean phospholipids and egg yolk phospholipids and so on.
As the glycolipid to be used, there can be mentioned, for example, cerebroside, sulfur-containing lipids (sulfa-tide), ganglioside and the like.
As the derived lipid, mention can be made of, for example, cholic acid, deoxycholic acid and so on. The amount thereof to be added ranges from about 10 ~mol to about 240 ~mol relative to 100 ~mol of phospholipid.
As the positive-charged lipid, any lipid having posi-tive charge can be used. As such positive-charged lipid, there can be mentioned, for example, saturated or unsaturated aliphatic amines having 12 - 22 carbon atoms, basic amino acids to which a fatty acid, cholesterol, etc. is bonded and .f~
the like. Specifically, mention can be made of oleyl~mine, stearylamine, etc. The amount thereof to be added ranges from about 20 ~mol to about 180 ~mol relative to 100 ~mol of phospholipid. Positive-charged lipid(s) is(are) used in a mixture with a phospholipid, and the proportion (molar ratio) of phospholipid to positive-charge lipid is in the range of from 9 to 2 to l to 18, preferably about 2 to l.
As the PG to be used in the present invention, there can be mentioned any prostanoic acid derivative possessing PG-like activity, which is exemplified by prostaglandins such as ( .g. PGFl, PGE2, PGE3), PGA (e.g. PGAl), PGF (e.g. PGF2) and PGD (e.g. PGD2), prostacyclins, thromboxanes, leuko-trienes, 6-keto-PGEl derivatives, carbacyclin derivatives, PGD2 derivatives and the like. The amount of PG to be added ranges from about l to 1000 ~g relative to 100 ~mol of phos-pholipid.
The production of the liposomes is outlined as follows:
By distilling off the solvent from a solution containing suitable lipid(s) and positive-charged lipid(s) (a solvent that does not degenerate lipid, e.g. chloroform, etc.), membranes of the lipids are prepared. To these membranes is added a solution containing PG, followed by vigorous shaking, and is preferably subjected to ultrasonication for dispersing the lipids homogeneously, to give a suspension of lipid thin membranes. As the solvent to be used for the solution con-taining PG, there can be used such a solvent as does not 2 ~
degenerate or decompose the liposome and is physiologically acceptable, which is exemplified by water (preferably, a buffer of pH 6 - 8, physiological saline, etc.), ethanol and the like.
In preparing the lipid membranes, there can be added as a stabilizing agent tocopherol (vi-tamine E), cholesterol, phosphatidic acid, dicetyl phosphate or a fatty acid such as palmitic acid.
~ lso, to the preparations of the present invention can be added, as a stabilizing agent, a macromolecular substance selected from albumin, dextran, a vinyl polymer, nonionic surfactant, gelatin and hydroxyethyl-starch.
Said macromolecular substances as a stabilizer may be entrapped in the gaps of the liposome together with the medicament, or can be added to or incorporated in the lipo-some preparation (namely, added or incorporated outside the liposome). It is needless to say that: said stabilizer may be incorporated both inside and outside the liposome. The amount of the stabilizer to be added is in the range of 0.5 to 10 parts by weight, preferably 1 to 5 parts by weight relative to 1 part by weight of the lipid.
In the present invention, it is preferable -to subject the mixture to ultrasonication. Then, for example, the particle diameter is adjusted to below 3 ~m and ultrasonica-tion is conducted under the conditions wherein the tempera-ture is 0C - 70C, preferably 35C - 45C, and the time is about 1 - 60 minutes.
By the above-mentioned procedure, there can be provided PG-containing particles of a diameter ranging from about 0.02 to about 0.1 ~Im. If desired, there can also be conducted molecular sieve procedure and gel filtration procedure for removing impurities or isolated substances.
The thus-obtained PG-containing liposome preparations contain PG in a proportion of over 0.1 part by weight rela-tive to 1 part by weight of the lipid, and the particle diameters are uniform and extremely fine. Therefore, they are preferable as medical preparations.
The entrapping rate of PG into the liposome is obtained by dissolving the prepared liposome in ethanol and analyzing the solution by liquid chromatography (gel filtration chroma-tography).
The liposome in which PG is entrapped can be recovered as precipitations. For example, the liposome can be reco-vered by subjecting the medium containing the liposome to ultracentrifugation. The liposome is, if desired, washed with a physiologically acceptable aqueous solution, and for-mulated into preparations in a pellet form or a suspension form. The formulation can be conducted in accordance with a method known widely in the field of pharmaceutical produc-tion. The preparations of the present invention can be provided as lyophilized preparations by freezing the liquid preparations, followed by drying under reduced pressure.
The present preparations can be used generally as an oral medicament or as an injectable medicament. The dosage ranges from 1 - 50 ~g on the basis of PG per an adult human and the preparations can be administered at the unit dosage of 0.02 - 1 ng/kg. The preparations are generally used by dissolving in or diluting with a physiologically acceptable aqueous solution, but they may also be formulated into tab-lets, capsules and eteric capsules by means of pharmaceutical production.
The present invention is more specifically described by the following experiment example and working examples, but they are not construed to limit the scope of the invention.
Example 1 In 5 ml of chloroform were dissolved 60 mg of marketed phosphatidyl choline originated from egg yolk, (hereinafter referred to as EPC) and 11 mg of oleyl amine, and a solution of 30 ~g of prostaglandin El dissolved in 100 ~1 of ethanol was added thereto. The mixture was put in a 25 ml-pear shape flask, from which the solvent was distilled off with rotary evaporator. A physiological saline containing 0.1 M phospho-ric acid (hereinafter referred to as PBS; pH 5.0)(1 ml) was added to the residue, and the mixture was subjected to sha-king, ultrasonication and centrifugation, whereafter the supernatant was filtered with a polycarbonate membrane filter of 0.2 ~m.
Example 2 In 10 ml of chloroform were dissolved 75 mg of marketed :' . ~ ''" ~ , ;
~ ' , phosphatidyl choline originated from egg yolk and 55 mg of oleylamine (hereinafter referred to as OAm), and a solution of 100 ~g of prostaglandin I2 dissolved in 200 ~1 of ethanol was added to the solution. A 25 ml-pear shape flask was charged with the mixture, from which the solvent was distil-led off with a rotary evaporator. To the residue was added 2 ml of a physiological saline containing 0.1 M phosphoric acid (pH 5.0), and the mixture was subjected to shaking, ultraso-nication and centrifugation, and then the supernatant was filtered with a polycarbonate membrane filter of 0.2 ~m.
Experiment Example The four kinds of liposomes as shown in the following Table 1 were prepared and evaluated Eor stability in neutral solvents.
Table 1 _ Liposome Composition of lipid Charge Form . .~ _ .
a EPC : OAm = 10 : O neutral SUV
b EPC : OAm = 10 : O neutral MLV large c EPC : OAm = 9 : 2 positive-charged SUV
d EPC : OAm = 8 : ~ positive-charged SUV
_ SUV stands for small unilamellar vesicle, MLV stands for multilamellar vesicle and MLV large stands for multi-lamellar vesicle with a large particle diameter.
Preparation of liposome containing PGEl labeled with tritium :. , In 0.5 ml of ethanol was dissolved about 5 mg of PGEl, and PGEl (25 ~Ci/0.25 ml, a 70% ethanol aqueous solution) labeled with tritium was mixed therewith.
In a 25 ml-pear shape flask were put a chloroform solution of EPC and the above-mentioned ethanol solution of PGEl in accordance with the compositions as shown in Table 2 respec-tively, and the solvent was distilled off with a rotary evaporator; to prepare lipid thin membranes.
Table 2 Liposome EPC OAm PGEl a 0.375 ml - 15 ~1 (100 ~mol) (100 ~g) b 0.375 ml - 15 ~1 (100 ~mol) (100 ~g) c 0.338 ml 0.132 ml 15 ~1 (90 ~mol) (20 ~mol) (100 ~g) d 0.300 ml 0.264 ml 15 ~1 (80 ~mol) (40 ~mol) (100 ~g) The lipid thin membranes were again dissolved ln chloro-form which was added thereto, and then the solvent was disti-lled off with a rotary evaporator to give lipid thin mem-branes. The procedures of dissolving in chloroform and pre-paring thin membranes were conducted repeatedly three times.
The flask of which the lipid thin membranes were formed on the inside wall was put in a desiccator, which was subjected to an hour's suction with a vacuum pump. In the flask was put 0.6 ml of PBS (pH 5.0), and the mixture was stirred with a Vortex mixer to give a lipid suspension (MLV).
The other suspensions of MLV except liposome b were made into SUV by sonication. The pear shape f lask containing the lipid suspension (MLV) was subjected to 20 minutes' sonication by a sonicator.
The suspension dispersed by sonication was diluted to a 2 ml-volume with PBS (pH 5.0), which was then subjected to 30 minutes' centrifugation at 15000 rpm. The supernatant was collected and filtered with a filter of 0.22 ~m.
Liposome b was diluted to a 2 ml-volume with PBS (pH 5.0), which was used as it was.
Test for stability in a neutral solvent To 0.45 ml of PBS (pH 7.4), 5~0 HSA (human serum albu-min)/PBS (pH 7.4) or 0.45 ml of rat plasma was added 0.05 ml of PGEl entrapped in liposome, and the mixture was thoroughly mixed. The cap of the tube filled with the mixture was closed, and the tube was soaked in a thermobath at 37C for incubation. After incubating for a certain time, gel filtra-tion with Sephacryl S-400 was conducted, and then the radio-activity, concentration of the phospholipid and absorbancy at 280 nm were measured.
Measurement of radioactivity , ~.
-:, , To 100 ~1 of the sampled liquid was added lO ml of liquid scintillater, and the mixture was stirred thoroughly.
Then, the radioactivity was measured.
Quantitative assay of phospholipid (PC) With the use of a reagent for measurement of phospholi-pid (enæymatic agent)(Manufactured by Denka Seiken), measure-ment was conducted.
Measurement of particle diameter The sample was diluted with a physiological saline, and measurement was conducted by light scattering method.
(Results of the Tests) Properties of liposome The pH of each of the liposomes was as shown in Table 3.
Since the pH of the liposome containing oleylamine (liposome d) was high, it was adjusted by addition of 1 N
hydrochloric acid.
Table 3 Liposome pH
a 4.hl b 4.78 c 5.73 + lN HCl ~0 ~ 4.61 d 6.07 + lN HCl 50 ~ 3.69 The mean particle diameters of the respective liposomes are shown in Table 4.
Table 4 Liposome Mean particle diameter (nm) , a 357.5 b 879.0 c 116.4 d 72.5 . .
~elease of PGEl from liposomes at pH 7.4 After the mixture of 50 ~1 of liposome with 950~1 of PBS (pH 7.4) was incubated at 37QC for 10 minutes, 500 ~1 of the mixture was subjected to gel filtration with PD-10 column e~uilibrated with PBS (pH 5~0). As the control, the liposome which was incubated with PBS (pH 5.0) was subjected to gel filtration in the same manner.
The profiles in the gel filtration of the respective liposomes are as shown in Fig. 1.
In the case of MLV (liposome b), the reason why the radioactivity of PGEl did not appear in the void fraction even at pH 5.0, is deemed to be that the particle diameters of MLV were too large to allow MLV to pass through the column.
In the case of the other liposomes incubated at pH 5.0, most of the radioactivity of PGEl appeared in the void fractions.
This shows that the behaviour of PGEl and that of liposome were identical.
On the other hand, in the case of liposomes a and b, , , , ' most of the radioactivity of PGEl disappeared from the void fractions when they were incubated at pH 7.4, and appearance of the radioactivity shifted to the lower molecular frac-tions. This indicates that PGEl was released from liposome.
In the case of the liposomes c and d, appearance of about half of the radioactivity of PGEl shifted to the lower mole-cular fractions, while the rest of the radioactivity stayed in the void fraction.
From the foregoing results, it is evident that making liposomes in multiple layers is not effective for preventing release of PGEl from liposomes at pH 7.4, but it is effective to add positive-charged llpid(s) to lipid membrane of lipo-somes.
Effects of albumin The behaviours in the presence of 5% HSA were examined for the liposomes a and d. Liposome ~0.05 ml) was mixed with 0.95 ml of PBS contalning 5% H',A (pH 5.0 and 7.4), and the mixture was incubated at 37C for 10 minutes. ~here-after, the mixture was subjected to gel filtration with Sephacryl S-400 column equilibrated with PBS (pH 5.0). The profile of the gel filtration is shown in Fig. 2.
The elution pattern of the liposome a which was incu-bated at pH 5.0 was one having two peaks in fractions 9 and 14. It is considered that fraction 9 is of PGEl adsorbed by HSA and fraction 14 is of free PGE1. In the case of the liposome a which was incubated at pH 7.4, there was no peak :.
in fraction 14.
In the case of the liposome d containing a positive-charged lipid (oleylamine), the rise of the peak in fraction 9 is not so sharp as that of the peak of liposome a and a portion of PGEl was eluted before fraction 9. This phenome-non was found both when incubated at pH 5.0 and pH 7.4.
From the foregoing results, it is considered that incor-porating positive-charged lipid(s) in liposome membranes is ef-fective for preventing release of PGEl from liposomes, even when albumin is present.
According to the present invention, release of PGs from liposomes can be suppressed. Especially, sufficient effects are achieved at pH in the visinity of neutral.
Therefore, the present invention can provide PG-contai-ning liposome preparations which are excellent in stability.
SPECIFICATION
Prostaglandin-containing Liposome Preparations FIELD OF THE INVENTION
This invention relates to prostaglandin-containing lipo-some preparations in which release of prostaglandin (herein-aEter referred to as PG) is suppressed.
BACKGRO~ND OF THE INVENTION
Since PGs possess various physiological actions such as vasodilating action, peripheral circulation-improving action, antihypertensive action, antilipolytic action and sodium diuretic action, they have been applied to pharmaceuticals.
In app]ying PGs to pharmaceuticals, there are problems that PGs metabolize into inactive substances easily in ]iving bodies and that they have little selectivity of focuses.
Therefore, preparations of PGs generally require fre-quent administrations, which is likely to give pain to pa-tients and also side-effects to tissues other than the target tissues. As a means to solve these problems, there have been provided preparations in which PGs are contained in a liposome.
Even in ~hese preparations of PGs-containing liposome, however, the stability thereof is not satisfactory and there is still a problem that PGs, which were once contained in the liposome get released.
SUMMARY OF THE INVENTION
The object of this invention is to provide PGs-containing liposome preparations in which release of PGs therefrom is suppressed.
The present inventors conducted various studies on the way to suppress release of PGs from liposome in preparations in which PGs are con-tained in liposome, and found that the problem could be solved by incorporating positive-charged lipid(s) in lipid membrane, which resulted in the comple-tion of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. la and lb show the profiles of gel filtration of the liposomes a - d, which indicate release of PGEl at pH
7.4. Fig. 2 shows the profiles of gel filtration of the liposomes a and d, which show release of PGEl in the presence of albumin.
DETAILED DESCRIPTION OF THE INVENTION
That is, this invention relates to prostaglandin-containing liposome preparations which are characteri~ed in that positive-charged lipid(s) is(are) incorporated in lipid membrane.
In this invention, fat corpuscles (liposome) have a structure of multiple concentric double layers, a single layer or multiple layers and are capable of entrapping va-rious proteins or other physiologically active substances in the gaps therein. The preparations of the present invention can be prepared by, for e~ample, bringing thin membranes made :' of phospholipid(s) or other kinds of lipids and positive-charged lipid(s) into contact with a solution containing PG and allowing PG to be entrapped in the lipid membrane and the gaps of the liposome.
As the lipid forming said liposomes, there may be mentioned phospholipids, glycolipids, derived lipids and the like. As the phospholipid, any phospholipid can be used as long as it is physiologically acceptable and can be matabo-lized. For example, use can be made of phosphatidyl choline, phosphatidyl serine, phosphatidic acid, phosphatidyl glyce-rin, phosphatidyl ethanolamine, phosphatidyl inositol, sphin-gomyelin, dicetyl phosphate, lysophosphatidyl choline (lyso-lecithine), mixtures thereof such as soybean phospholipids and egg yolk phospholipids and so on.
As the glycolipid to be used, there can be mentioned, for example, cerebroside, sulfur-containing lipids (sulfa-tide), ganglioside and the like.
As the derived lipid, mention can be made of, for example, cholic acid, deoxycholic acid and so on. The amount thereof to be added ranges from about 10 ~mol to about 240 ~mol relative to 100 ~mol of phospholipid.
As the positive-charged lipid, any lipid having posi-tive charge can be used. As such positive-charged lipid, there can be mentioned, for example, saturated or unsaturated aliphatic amines having 12 - 22 carbon atoms, basic amino acids to which a fatty acid, cholesterol, etc. is bonded and .f~
the like. Specifically, mention can be made of oleyl~mine, stearylamine, etc. The amount thereof to be added ranges from about 20 ~mol to about 180 ~mol relative to 100 ~mol of phospholipid. Positive-charged lipid(s) is(are) used in a mixture with a phospholipid, and the proportion (molar ratio) of phospholipid to positive-charge lipid is in the range of from 9 to 2 to l to 18, preferably about 2 to l.
As the PG to be used in the present invention, there can be mentioned any prostanoic acid derivative possessing PG-like activity, which is exemplified by prostaglandins such as ( .g. PGFl, PGE2, PGE3), PGA (e.g. PGAl), PGF (e.g. PGF2) and PGD (e.g. PGD2), prostacyclins, thromboxanes, leuko-trienes, 6-keto-PGEl derivatives, carbacyclin derivatives, PGD2 derivatives and the like. The amount of PG to be added ranges from about l to 1000 ~g relative to 100 ~mol of phos-pholipid.
The production of the liposomes is outlined as follows:
By distilling off the solvent from a solution containing suitable lipid(s) and positive-charged lipid(s) (a solvent that does not degenerate lipid, e.g. chloroform, etc.), membranes of the lipids are prepared. To these membranes is added a solution containing PG, followed by vigorous shaking, and is preferably subjected to ultrasonication for dispersing the lipids homogeneously, to give a suspension of lipid thin membranes. As the solvent to be used for the solution con-taining PG, there can be used such a solvent as does not 2 ~
degenerate or decompose the liposome and is physiologically acceptable, which is exemplified by water (preferably, a buffer of pH 6 - 8, physiological saline, etc.), ethanol and the like.
In preparing the lipid membranes, there can be added as a stabilizing agent tocopherol (vi-tamine E), cholesterol, phosphatidic acid, dicetyl phosphate or a fatty acid such as palmitic acid.
~ lso, to the preparations of the present invention can be added, as a stabilizing agent, a macromolecular substance selected from albumin, dextran, a vinyl polymer, nonionic surfactant, gelatin and hydroxyethyl-starch.
Said macromolecular substances as a stabilizer may be entrapped in the gaps of the liposome together with the medicament, or can be added to or incorporated in the lipo-some preparation (namely, added or incorporated outside the liposome). It is needless to say that: said stabilizer may be incorporated both inside and outside the liposome. The amount of the stabilizer to be added is in the range of 0.5 to 10 parts by weight, preferably 1 to 5 parts by weight relative to 1 part by weight of the lipid.
In the present invention, it is preferable -to subject the mixture to ultrasonication. Then, for example, the particle diameter is adjusted to below 3 ~m and ultrasonica-tion is conducted under the conditions wherein the tempera-ture is 0C - 70C, preferably 35C - 45C, and the time is about 1 - 60 minutes.
By the above-mentioned procedure, there can be provided PG-containing particles of a diameter ranging from about 0.02 to about 0.1 ~Im. If desired, there can also be conducted molecular sieve procedure and gel filtration procedure for removing impurities or isolated substances.
The thus-obtained PG-containing liposome preparations contain PG in a proportion of over 0.1 part by weight rela-tive to 1 part by weight of the lipid, and the particle diameters are uniform and extremely fine. Therefore, they are preferable as medical preparations.
The entrapping rate of PG into the liposome is obtained by dissolving the prepared liposome in ethanol and analyzing the solution by liquid chromatography (gel filtration chroma-tography).
The liposome in which PG is entrapped can be recovered as precipitations. For example, the liposome can be reco-vered by subjecting the medium containing the liposome to ultracentrifugation. The liposome is, if desired, washed with a physiologically acceptable aqueous solution, and for-mulated into preparations in a pellet form or a suspension form. The formulation can be conducted in accordance with a method known widely in the field of pharmaceutical produc-tion. The preparations of the present invention can be provided as lyophilized preparations by freezing the liquid preparations, followed by drying under reduced pressure.
The present preparations can be used generally as an oral medicament or as an injectable medicament. The dosage ranges from 1 - 50 ~g on the basis of PG per an adult human and the preparations can be administered at the unit dosage of 0.02 - 1 ng/kg. The preparations are generally used by dissolving in or diluting with a physiologically acceptable aqueous solution, but they may also be formulated into tab-lets, capsules and eteric capsules by means of pharmaceutical production.
The present invention is more specifically described by the following experiment example and working examples, but they are not construed to limit the scope of the invention.
Example 1 In 5 ml of chloroform were dissolved 60 mg of marketed phosphatidyl choline originated from egg yolk, (hereinafter referred to as EPC) and 11 mg of oleyl amine, and a solution of 30 ~g of prostaglandin El dissolved in 100 ~1 of ethanol was added thereto. The mixture was put in a 25 ml-pear shape flask, from which the solvent was distilled off with rotary evaporator. A physiological saline containing 0.1 M phospho-ric acid (hereinafter referred to as PBS; pH 5.0)(1 ml) was added to the residue, and the mixture was subjected to sha-king, ultrasonication and centrifugation, whereafter the supernatant was filtered with a polycarbonate membrane filter of 0.2 ~m.
Example 2 In 10 ml of chloroform were dissolved 75 mg of marketed :' . ~ ''" ~ , ;
~ ' , phosphatidyl choline originated from egg yolk and 55 mg of oleylamine (hereinafter referred to as OAm), and a solution of 100 ~g of prostaglandin I2 dissolved in 200 ~1 of ethanol was added to the solution. A 25 ml-pear shape flask was charged with the mixture, from which the solvent was distil-led off with a rotary evaporator. To the residue was added 2 ml of a physiological saline containing 0.1 M phosphoric acid (pH 5.0), and the mixture was subjected to shaking, ultraso-nication and centrifugation, and then the supernatant was filtered with a polycarbonate membrane filter of 0.2 ~m.
Experiment Example The four kinds of liposomes as shown in the following Table 1 were prepared and evaluated Eor stability in neutral solvents.
Table 1 _ Liposome Composition of lipid Charge Form . .~ _ .
a EPC : OAm = 10 : O neutral SUV
b EPC : OAm = 10 : O neutral MLV large c EPC : OAm = 9 : 2 positive-charged SUV
d EPC : OAm = 8 : ~ positive-charged SUV
_ SUV stands for small unilamellar vesicle, MLV stands for multilamellar vesicle and MLV large stands for multi-lamellar vesicle with a large particle diameter.
Preparation of liposome containing PGEl labeled with tritium :. , In 0.5 ml of ethanol was dissolved about 5 mg of PGEl, and PGEl (25 ~Ci/0.25 ml, a 70% ethanol aqueous solution) labeled with tritium was mixed therewith.
In a 25 ml-pear shape flask were put a chloroform solution of EPC and the above-mentioned ethanol solution of PGEl in accordance with the compositions as shown in Table 2 respec-tively, and the solvent was distilled off with a rotary evaporator; to prepare lipid thin membranes.
Table 2 Liposome EPC OAm PGEl a 0.375 ml - 15 ~1 (100 ~mol) (100 ~g) b 0.375 ml - 15 ~1 (100 ~mol) (100 ~g) c 0.338 ml 0.132 ml 15 ~1 (90 ~mol) (20 ~mol) (100 ~g) d 0.300 ml 0.264 ml 15 ~1 (80 ~mol) (40 ~mol) (100 ~g) The lipid thin membranes were again dissolved ln chloro-form which was added thereto, and then the solvent was disti-lled off with a rotary evaporator to give lipid thin mem-branes. The procedures of dissolving in chloroform and pre-paring thin membranes were conducted repeatedly three times.
The flask of which the lipid thin membranes were formed on the inside wall was put in a desiccator, which was subjected to an hour's suction with a vacuum pump. In the flask was put 0.6 ml of PBS (pH 5.0), and the mixture was stirred with a Vortex mixer to give a lipid suspension (MLV).
The other suspensions of MLV except liposome b were made into SUV by sonication. The pear shape f lask containing the lipid suspension (MLV) was subjected to 20 minutes' sonication by a sonicator.
The suspension dispersed by sonication was diluted to a 2 ml-volume with PBS (pH 5.0), which was then subjected to 30 minutes' centrifugation at 15000 rpm. The supernatant was collected and filtered with a filter of 0.22 ~m.
Liposome b was diluted to a 2 ml-volume with PBS (pH 5.0), which was used as it was.
Test for stability in a neutral solvent To 0.45 ml of PBS (pH 7.4), 5~0 HSA (human serum albu-min)/PBS (pH 7.4) or 0.45 ml of rat plasma was added 0.05 ml of PGEl entrapped in liposome, and the mixture was thoroughly mixed. The cap of the tube filled with the mixture was closed, and the tube was soaked in a thermobath at 37C for incubation. After incubating for a certain time, gel filtra-tion with Sephacryl S-400 was conducted, and then the radio-activity, concentration of the phospholipid and absorbancy at 280 nm were measured.
Measurement of radioactivity , ~.
-:, , To 100 ~1 of the sampled liquid was added lO ml of liquid scintillater, and the mixture was stirred thoroughly.
Then, the radioactivity was measured.
Quantitative assay of phospholipid (PC) With the use of a reagent for measurement of phospholi-pid (enæymatic agent)(Manufactured by Denka Seiken), measure-ment was conducted.
Measurement of particle diameter The sample was diluted with a physiological saline, and measurement was conducted by light scattering method.
(Results of the Tests) Properties of liposome The pH of each of the liposomes was as shown in Table 3.
Since the pH of the liposome containing oleylamine (liposome d) was high, it was adjusted by addition of 1 N
hydrochloric acid.
Table 3 Liposome pH
a 4.hl b 4.78 c 5.73 + lN HCl ~0 ~ 4.61 d 6.07 + lN HCl 50 ~ 3.69 The mean particle diameters of the respective liposomes are shown in Table 4.
Table 4 Liposome Mean particle diameter (nm) , a 357.5 b 879.0 c 116.4 d 72.5 . .
~elease of PGEl from liposomes at pH 7.4 After the mixture of 50 ~1 of liposome with 950~1 of PBS (pH 7.4) was incubated at 37QC for 10 minutes, 500 ~1 of the mixture was subjected to gel filtration with PD-10 column e~uilibrated with PBS (pH 5~0). As the control, the liposome which was incubated with PBS (pH 5.0) was subjected to gel filtration in the same manner.
The profiles in the gel filtration of the respective liposomes are as shown in Fig. 1.
In the case of MLV (liposome b), the reason why the radioactivity of PGEl did not appear in the void fraction even at pH 5.0, is deemed to be that the particle diameters of MLV were too large to allow MLV to pass through the column.
In the case of the other liposomes incubated at pH 5.0, most of the radioactivity of PGEl appeared in the void fractions.
This shows that the behaviour of PGEl and that of liposome were identical.
On the other hand, in the case of liposomes a and b, , , , ' most of the radioactivity of PGEl disappeared from the void fractions when they were incubated at pH 7.4, and appearance of the radioactivity shifted to the lower molecular frac-tions. This indicates that PGEl was released from liposome.
In the case of the liposomes c and d, appearance of about half of the radioactivity of PGEl shifted to the lower mole-cular fractions, while the rest of the radioactivity stayed in the void fraction.
From the foregoing results, it is evident that making liposomes in multiple layers is not effective for preventing release of PGEl from liposomes at pH 7.4, but it is effective to add positive-charged llpid(s) to lipid membrane of lipo-somes.
Effects of albumin The behaviours in the presence of 5% HSA were examined for the liposomes a and d. Liposome ~0.05 ml) was mixed with 0.95 ml of PBS contalning 5% H',A (pH 5.0 and 7.4), and the mixture was incubated at 37C for 10 minutes. ~here-after, the mixture was subjected to gel filtration with Sephacryl S-400 column equilibrated with PBS (pH 5.0). The profile of the gel filtration is shown in Fig. 2.
The elution pattern of the liposome a which was incu-bated at pH 5.0 was one having two peaks in fractions 9 and 14. It is considered that fraction 9 is of PGEl adsorbed by HSA and fraction 14 is of free PGE1. In the case of the liposome a which was incubated at pH 7.4, there was no peak :.
in fraction 14.
In the case of the liposome d containing a positive-charged lipid (oleylamine), the rise of the peak in fraction 9 is not so sharp as that of the peak of liposome a and a portion of PGEl was eluted before fraction 9. This phenome-non was found both when incubated at pH 5.0 and pH 7.4.
From the foregoing results, it is considered that incor-porating positive-charged lipid(s) in liposome membranes is ef-fective for preventing release of PGEl from liposomes, even when albumin is present.
According to the present invention, release of PGs from liposomes can be suppressed. Especially, sufficient effects are achieved at pH in the visinity of neutral.
Therefore, the present invention can provide PG-contai-ning liposome preparations which are excellent in stability.
Claims (10)
1. A prostaglandin-containing liposome preparation, charac-terized in that positive-charged lipid(s) is(are) incorpo-rated in lipid thin membrane therein.
2. A prostaglandin-containing liposome preparation as claimed in Claim 1 wherein the lipid(s) forming the liposomes is (are) selected from the group consisting of phospholipids, glycolipids and derived lipids.
3. A prostaglandin-containing liposome preparation as claimed in Claim 1, characterized in that the positive-charged lipid(s) is(are) aliphatic amines having 12 - 22 carbon atoms or basic amino acids to which a fatty acid or cholesterol is bonded.
4. A prostaglandin-containing liposome preparation as claimed in Claim 1, characterized in that the positive-charged lipid(s) comprise(s) at least oleylamine or stearyl-amine.
5. A prostaglandin-containing liposome preparation as claimed in Claim 1 wherein the proportion (molar ratio) of phospholipid to positive-charge lipid is in the range of from 9 to 2 to 1 to 18.
6. A prostaglandin-containing liposome preparation as claimed in Claim 1 wherein the prostaglandin is selected from among the group consisting of PGE, PGA, PGF and PGD.
7. A prostaglandin-containing liposome preparation as claimed in Claim 1 wherein the prostaglandin is selected from among the group consisting of PGE1, PGE2 and PGE3.
8. A prostaglandin-containing liposome preparation as claimed in Claim 1 wherein the amount of prostaglandin to be added ranges from about 1 to 1000 µg relative to 100 µmol of lipid.
9. A prostaglandin-containing liposome preparation as claimed in Claim 1 wherein the particle diameter is in the range of about 0,02 to about 0.1 µm.
10. A prostagiandin-containing liposome preparation as claimed in Claim 1, which contains prostaglandin in a proportion of over 0.1 part by weight relative to 1 part by weight of the lipid.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1232479A JPH0395118A (en) | 1989-09-07 | 1989-09-07 | Prostaglandin-containing liposome preparation |
| JP232479/1989 | 1989-09-07 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2024804A1 true CA2024804A1 (en) | 1991-03-08 |
Family
ID=16939951
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002024804A Abandoned CA2024804A1 (en) | 1989-09-07 | 1990-09-06 | Prostaglandin-containing liposome preparations |
Country Status (3)
| Country | Link |
|---|---|
| EP (1) | EP0416527A3 (en) |
| JP (1) | JPH0395118A (en) |
| CA (1) | CA2024804A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5853755A (en) * | 1993-07-28 | 1998-12-29 | Pharmaderm Laboratories Ltd. | Biphasic multilamellar lipid vesicles |
Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5811118A (en) * | 1987-05-22 | 1998-09-22 | The Liposome Company, Inc. | Methods of treatment using unilamellar liposomal arachidonic acid metabolite formulations |
| US5925375A (en) * | 1987-05-22 | 1999-07-20 | The Liposome Company, Inc. | Therapeutic use of multilamellar liposomal prostaglandin formulations |
| US5552155A (en) * | 1992-12-04 | 1996-09-03 | The Liposome Company, Inc. | Fusogenic lipsomes and methods for making and using same |
| IL107471A (en) * | 1993-11-02 | 1997-09-30 | Yissum Res Dev Co | Pharmaceutical preparation comprising a self- emulsifying formulation and process for its production |
| ATE191141T1 (en) * | 1993-11-04 | 2000-04-15 | Liposome Co Inc | TREATMENT METHODS USING UNILAMELLAR LIPOSOMAL ARACHIDDONIC ACID METABOLITE FORMULATIONS |
| EP0758883B1 (en) * | 1994-04-12 | 2003-03-19 | The Liposome Company, Inc. | Fusogenic liposomes and methods of making and using same |
| AU692255B2 (en) * | 1995-04-24 | 1998-06-04 | Yissum Research Development Company Of The Hebrew University Of Jerusalem | Self-emulsifiable formulation producing an oil-in-water emulsion |
| US5718917A (en) * | 1995-12-15 | 1998-02-17 | Harvard Scientific Corporation | PGE-1 containing lyophilized liposomes for use in the treatment of erectile dysfunction |
| US5837283A (en) | 1997-03-12 | 1998-11-17 | The Regents Of The University Of California | Cationic lipid compositions targeting angiogenic endothelial cells |
| US7112338B2 (en) | 1997-03-12 | 2006-09-26 | The Regents Of The University Of California | Cationic liposome delivery of taxanes to angiogenic blood vessels |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4692433A (en) * | 1983-10-12 | 1987-09-08 | The Regents Of The University Of California | Method and composition for regulating serum calcium levels of mammals |
| AU617678B2 (en) * | 1987-05-22 | 1991-12-05 | Liposome Company, Inc., The | Prostaglandin-lipid formulations |
-
1989
- 1989-09-07 JP JP1232479A patent/JPH0395118A/en active Pending
-
1990
- 1990-09-04 EP EP19900116948 patent/EP0416527A3/en not_active Withdrawn
- 1990-09-06 CA CA002024804A patent/CA2024804A1/en not_active Abandoned
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5853755A (en) * | 1993-07-28 | 1998-12-29 | Pharmaderm Laboratories Ltd. | Biphasic multilamellar lipid vesicles |
| US5993851A (en) * | 1993-07-28 | 1999-11-30 | Pharmaderm Laboratories, Ltd. | Method for preparing biphasic multilamellar lipid vesicles |
Also Published As
| Publication number | Publication date |
|---|---|
| EP0416527A2 (en) | 1991-03-13 |
| JPH0395118A (en) | 1991-04-19 |
| EP0416527A3 (en) | 1992-10-14 |
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